The biobleaching potential of laccase produced from mandarin peelings: Impetus for a circular bio-based economy in textile biofinishing

2.1 The inoculum

A laccase-producing bacterial strain coded Hb28a, isolated from lithospheric microbiota under xeric conditions, i.e. rock scrapings from a semi-arid region of South Africa (32°78′59″S and 26°84′85″E), was employed in this study. It was selectively enriched by orbital incubation (140 rpm at 30 °C) for 7 days in physiological saline supplemented with 1g/L kraft lignin, 0.02g/L of potassium hydrogen phthalate (PHP) and vanillin. Thereafter, fivefold serial dilutions of 1 mL aliquots in physiological saline was performed under aseptic conditions, where 500 µL volumes were spread on a mineral salt medium comprising (g/L): agar; 13.0, NaNO₃; 2.6, K₂HPO₄; 0.4, KH₂PO₄; 0.6, MgSO₄·7H₂O; 0.5, NaCl; 0.5, which was supplemented with 1 g/L kraft lignin, 0.02 g/L PHP and 0.02 g/L vanillin, and then incubated at 30 °C for 7 days. It was further screened for laccase activity as described by Unuofin et al. (2019d), using 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6 dimethoxyphenol (DMP), guaiacol, 1-naphthol, syringaldazine and potassium ferrocyanoferrate (PFC). The bacterial strain was selected on the basis of its oxidation of the aforementioned chemical substrates and was further identified using 16S rRNA sequence analysis as the pseudomonad, Pseudomonas aeruginosa HRJ16 (used henceforth as HRJ16) with accession number MF073259 in GenBank, National Center for Biotechnology Information (NCBI). Appropriate portions of axenic culture were maintained as glycerol stock at −80 °C or as working stock at 4 °C. Prior to further use, working stock culture was gradually adapted to room temperature before it was passed to freshly prepared broth, where it was standardized to OD_{600 nm} of 0.8 after 18 h of incubation.

2.2 Screening, process optimization and laccase production

A one variable at a time (OVAT) was used to screen for significant cultural and nutritional factors, whereas statistical optimization (response surface methodology) was adopted to optimize laccase production. In this manner, the pH, temperature, agitation, carbon sources, nitrogen sources, agroindustrial residues, and aromatic and inorganic inducers, independent of other factors in the experiment. One percent (1 % w/v) carbohydrates (glucose, trehalose, xylose, cellobiose, fructose, galactose), and an aliphatic alcohol (glycerol), were varied as carbon sources, while 0.2 % w/v nitrogenous sources (NaNO₃, KNO₃, NH₄NO₃, l-asparagine, yeast extract, tryptone), and 0.05 % w/v inducers (CoCl₂, CuSO₄, ferulic acid, acetaminophen, 4-nitrophenol, vanillic acid, 2,5-xylidine) were screened for their respective contributions to laccase production. The impacts of agro-industrial residues, such as, maize stover (MS), wheat bran (WB), grape stalks (GS) and mandarin peelings (MP) were assessed. For response surface methodology (RSM), four cardinal independent variables observed from OVAT were inputted into a central composite design (CCD) generated by Design-Expert (Stat-Ease, Inc., Minneapolis, USA, version 10.0.3). The 30 experimental (3-level-4-factorial) runs produced by this algorithm were reproduced empirically in 100 mL Schott bottles containing 20 mL mineral salt medium (MSM) (stated infra) and inoculated with 400 µL standardized culture (1.53 × 10⁸ cfu/mL). Thereafter, results were fit to a quadratic model Eq (1), which was further explored to understand and optimize the interactions of different variables.

Y = Responses.

K = Total number of independent factors.

Β₀  = Intercept.

β_i, β_ii, and β_ij = Coefficient values for linear, quadratic and interaction effects respectively.and x_j indicate coded levels for independent variables.

All experiments were conducted in triplicates and bottles were incubated at 30 °C for 96 h except otherwise stated. The aforementioned MSM comprised (g/L pH citrate): KH₂PO₄; 0.514, K₂HPO₄; 0.32, KNaC₄H₄O₆·4H₂O; 0.32, NaCl; 0.08, MnSO·H₂O; 0.032, MgSO₄·7H₂O; 0.192, CaCl₂·2H₂O; 0.008, CuSO₄·5H₂O; 0.0008, FeCl₃·7H₂O; 0.0008, ZnSO₄; 0.0008. (Sigma-Aldrich, South Africa). Bulk fermentation for laccase production entailed supplementing MSM with mandarin peelings; 11.25 g/L, NaNO₃; 0.375 g/L, 0.05 % w/v acetaminophen and orbital conditions of 100 rpm at 30 °C based on RSM numerical optimization outcomes. Aqueous crude laccase extracts were separated by cold centrifugation (4 °C) at 20,124 × g for 12 min. Laccase activity was measured as the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in a reaction comprising 50 µL appropriately diluted laccase, phosphate buffered (100 mM, pH 6) 2 mM ABTS, and 40 µL of 20 % v/v trichloroacetic acid (TCA) to terminate a 10 min reaction at 30 °C. The colorimetric output of ABTS oxidation was visualized at 420 nm (ε = 36,000 M⁻¹ cm⁻¹) with the aid of a SynergyMX 96-well microtitre plate reader (BioTek Instruments). One unit of laccase was extrapolated as the concentration that successfully oxidized 1 µmol of ABTS per min.

2.3 Biochemical characterization of laccase and molecular signature

Certain biochemical properties of crude HRJ16 laccase were evaluated in buffered solutions, according to Unuofin (2020b). Temperature optima was assessed at ranges between 0 °C and 90 °C by incubation in phosphate buffer (pH 6, 100 mM) for 30 mins, whereas temperature stability measurements were taken over 1440 mins for 40 °C −90 °C. Optimal pH was evaluated at 30 °C for 30 mins, while incubated in buffer ranging from pH 3–11 (citrate, phosphate and carbonate-bicarbonate) and stability measurements were observed for the same buffers over 455 mins. Effect of different concentrations of some metal ions (Mn²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Ba²⁺, Zn²⁺, Mg²⁺, Co²⁺), surfactants (SDS, Tween 20), Chelator (EDTA) solvent (DMSO), halides (F^-, Cl^-) were assessed after 30 min preincubation with crude laccase. Correspondingly, substrate specificity studies were conducted in replicates, as described by the aforesaid study. Laccase-catalyzed oxidations of ABTS, guaiacol, syringaldazine (SGZ), α-naphthol and PFC in a phosphate buffer (100 mM; pH6) were monitored and estimated at the respective wavelengths and substrate extinction coefficients: 420 nm (ε = 36,000 M⁻¹ cm⁻¹), 470 nm (ε = 26,600 M⁻¹ cm⁻¹), 530 nm (ε = 65,000 M⁻¹ cm⁻¹), 520 nm (ε = 57,490 M⁻¹ cm⁻¹) and 420 nm (ε = 1023 M⁻¹ cm⁻¹). Briefly, 2 mM substrates in pH 6 potassium phosphate buffer were incubated at room temperature with 100 μL crude laccase, except for ABTS which was reacted with 60 μL of appropriately diluted laccase at 30 °C, allowed to react for 10 min and thereafter stopped with 40 μL 20 % TCA. Genomic DNA of HRJ16 was extracted according to the method of Unuofin et al. (2019e). Pellets of axenic HRJ16 were vortexed in nuclease free water and then boiled in an AccuBlock Digital dry bath (TECHNE, LAsec SA) at 100 °C for 10 mins and then centrifuged at 15,155 × g for 5 mins. The resulting supernatant contained the genomic DNA and was aseptically withdrawn for PCR. The PCR conditions for DNA amplification, with primers used, and the procedures for gel electrophoresis and visualization of target bands have been previously described (Unuofin et al., 2019e). The PCR mixture contained 5 μL template DNA, 12.5 μL 2 × OneTaq PCR MasterMix (Biolabs, South Africa), 1 μL (10 μM) of each forward and reverse primer, and was adjusted to a total volume of 25 μL with nuclease-free sterile water (Life Science). PCR amplification was run on a thermal cycler (G-STORM, UK) and the amplicons were run on 1.7 % w/v agarose gel (Merck, SA) in a Submarine Electrophoresis System (Mupid-One, Takara, ADVANCE Co., ltd. Japan) for 45 mins at 100 V, and thereafter had their bands visualized with the aid of ethidium bromide stains (Sigma-Aldrich, South Africa) in a transilluminator (ALLIANCE 4.7, France) with the UVITEC Cambridge software.

2.4 Dye decolorization and denim bioscouring

Fifty milliliters (50 mL) phosphate buffered homogenates (50 mM, pH 6) comprising 100 mg synthetic dyes and their respective colour index numbers (CI): Azure B 52,010 (AB), Malachite Green 42,000 (MG), Reactive Blue 4 61,205 (RB), Methyl Orange 13,025 (MO), Congo Red 22,120 (CR), and Brilliant Blue G 42,655 (BB) were prepared and incubated with HRJ16 laccase. Control treatments contained the decolourization mixtures without the crude laccases. Two milliliters (2 mL) of reaction mixture comprised 50 µL of crude laccase (416.4 U/mL) and 1950 µL dye solution in hermetically sealed tubes. All the decolourizing reactions were carried out at 30 °C and absorbances were intermittently recorded for over 80 h with the aid of a Synergy MX Microplate reader (BioTek^TM) at the respective wavelengths (Unuofin, 2020b). Thereafter, their decolorization efficiencies were calculated as follows: $$\begin{array}{c}A=-{\log }_{10}\frac{I}{I_o}={\log }_{10}\frac{I_o}{I}\\ \frac{I}{I_o}={10}^{-A}\Rightarrow \frac{I_o}{I}=\frac{1}{{10}^{-A}}\\ \frac{I}{I_o}={10}^{-A}=R\\ \%R=R\times 100\end{array}$$

Where, A_initial is the initial absorbance, and A_observed is the observed absorbance.

The biobleaching potential of HRJ16 laccase was assayed on an indigo-dyed denim garment. Square portions (4 cm × 4 cm) of the fabric were snipped out and thereafter exposed to two treatments which comprised: (i) crude laccase and (ii) crude laccase + 2 mM ABTS. However, a treatment comprising 2 mM ABTS only was adopted as control. The setups were incubated in phosphate buffer (pH6; 100 mM) at 30 °C, 120 rpm for a period of 6 h. Thereafter, each swatch was gently rinsed in sterile deionized water to minimize abrasion of fabric during dye wash-down. The rinsed pieces were thereafter air-dried and observed under the dissection microscope (×30) (KYOWA TOKYO No.751252). Furthermore, their absorbance coefficients were evaluated at 300 nm to 900 nm wavelength range with a spectrophotometer (VWR® UV-3000PC), from which % reflectance was extrapolated through the following equation:

2.5 Data analysis

Results of replicates were pooled and expressed as mean ± standard deviation (SD) using Microsoft Excel Spreadsheet. Data were subsequently subjected to one-way analysis of variance (ANOVA) and the least significant difference was carried out. Significance was identified at P ≤ 0.05.

3.1 Biochemical and molecular features of HRJ16 laccase

Optimal reaction conditions, such as pH 8 and 70 °C were recorded for HRJ16 laccases (Fig. 3a and Fig. 3b). Correspondingly, the relative activity profile of the aforementioned parameters tested portrayed the enzyme as a thermophilic, neutral to alkaline-enabled protein. A spore laccase of Bacillus amyloliquefaciens had an optimum pH and temperature of 7.5 and 60 °C (El-Bendary et al., 2021); whereas Edoamodu and Nwodo (2021) recorded pH 6 and 80 °C as optimal regime for laccase activity in Enterobacter asburiae ES1. Our statistics for laccase activity were matched by an impressive stability record at pH 3–11 over 455 mins as well as 40 °C – 90 °C, over 400 mins (Fig. 4a and Fig. 4b); albeit incidental evaluations at 1140 and 1440 mins revealed detectable activities of at least ca. 60 % residual laccase. A similar trend was observed in an Achromobacter specie (Unuofin et al., 2019e); however, stability properties exhibited thereof were at least ca. 100 % for all pH regimes over 455 mins and at least ca. 92 % for temperatures spanning 40 °C and 80 °C, over 1440 mins, respectively. In comparison, Alcaligenes faecalis XF1 laccase achieved at least ca. 80 % stability at 60–80 °C over 120 min, while a 10-day stability of at least 80 % was observed at pH 6–8 (Mehandia et al., 2020). Essentially, pH and temperature extremes are what characterizes most industrial reaction conditions. Subsequently, these parameters are for the most part unchanged during toxic waste discharge up till certain distances downstream from initial contact point. Knowing these conditions hardly change, it is necessary to appropriately employ thermotolerant green catalysts as bacterial laccases to treat wastes, prior to discharge. On incubation with different concentrations of some cations, chelator and surfactant (Fig. 4c), HRJ16 laccase exhibited remarkable stability, presenting residual activities that insinuated inducement especially notable with Mn²⁺ (2.5 mM and 7 mM), Cu²⁺ (2.5 mM and 5 mM), and Co²⁺ (1 mM). In a study by Motamedi and co-authors (2021) on a metagenome derived laccase (PersiLac2), 10 mM concentrations of K⁺, Li²⁺, Mg²⁺, Fe²⁺, Zn²⁺, Al²⁺, Ni²⁺, Mn²⁺ elicited residual activities beyond 250 %, while 600 mM and 800 mM Cu²⁺ stimulated laccase activity beyond 200 %. However, the major highlight was the overexpression of activity due to the comparative stimulation of different concentrations of chelator and surfactant (Fig. 4c). These particular outcomes coincide with a previous study, where all concentrations of Mn²⁺, Fe³⁺, Ba²⁺, Cu²⁺, EDTA and SDS effected progression of residual activity beyond the control (Unuofin, 2021). However, this positive stimulation was not observed for Motamedi et al. (2021), who recorded noticeable inhibitory activities of some surfactants, albeit at higher concentrations (10 mM and 25 mM). Our results further demonstrate the capacity of HRJ16 laccase as an essential green chemical for commercial applications where heavy metals, surfactants or chelators would be a barrier. Particularly, the laccase was able to thrive in the presence of the chelator (EDTA), which will have otherwise impeded its catalytic proficiency. Other biochemical characteristics portrayed by HRJ16 laccase, which are worthy of mention include tolerance of high concentrations of NaCl, DMSO and Tween 20 (Fig. 4d) as well as its remarkable inducement by the most potent inhibitory halide NaF (Fig. 4e). Similarly, DMSO at 5 %, 10 % and 20 % v/v concentrations elicited a residual activity beyond 100 %, while 1 M and 2 M concentrations of NaCl correspondingly stimulated residual activities beyond 100 % (Motamedi et al., 2021). In another study, the non-ionic surfactant, Tween 20 recorded at least 80 % residual activity at 0.1 mM, 1.0 mM and 5.0 mM (Sondhi et al., 2021), while the inhibitory activity of NaF was expressed at 10 mM thereby permitting about 1 % residual activity (Sondhi et al., 2021). From the extrapolation of a kinetic plot (R² = 0.982) (Fig S2), HRJ16 laccase was presumed to elicit a K_m of 2.6 µM, Kcat of 1.85 × 10⁴ S^-1 and a specificity constant of 6.94 × 10³ µM⁻¹ s⁻¹ (Table 2). This indicates a strong affinity HRJ16 laccase has for the ABTS assay substrate as well as the spontaneity and rapidity at which substrate molecules are being transformed to products or intermediates. A further look at Table 2 portrays its comparison to other laccases. This trend had been earlier detected during assessment of substrates for laccase catalytic compatibility (Fig. 5), where ABTS and PFC were more readily transformed than their organic confrére. Moreover, they may serve as excellent electron shuttles to initiate the catalytic oxidation of larger or unreactive substrates, hence affording laccase a wide range of unorthodox applications in environmental biotechnology.

Close

Fig. 3a

Effect of pH regime on HRJ16 laccase activity.

Export to PPT

Close

Fig. 3b

Effect of temperature profile on HRJ16 laccase activity.

Export to PPT

Close

Fig. 4a

pH stability exhibited by HRJ16 laccase at 30 °C.

Export to PPT

Close

Fig. 4b

Thermostable behaviour of HRJ16 laccase at pH 6 (100 mM phosphate buffer).

Export to PPT

Close

Fig. 4c

HRJ16 laccase response to varying concentrations of cations, chelator and surfactant.

Export to PPT

Close

Fig. 4d

Response of HRJ16 laccase to halide (NaCl), surfactant (Tween 20) and solvent (DMSO).

Export to PPT

Close

Fig. 4e

Effect of increasing NaF concentrations on HRJ16 laccase stability.

Export to PPT

Close

Fig. 5

Substrate specificity of HRJ16 laccase.

Export to PPT

The molecular snapshot survey of laccase producing HRJ16 used in this study revealed it to have multiple homologous genes (Fig. 6), which might be suggestive of their participation in diverse morpho-functional activities. In essence, such genes might comprise constitutive, which are regarded as housekeeping genes, and inducible loci, which are only expressed under certain environmental stresses just as was corroborated by Yan et al. (2019). Multiple laccase genes have been reported in plants (Arcuri et al., 2020; Liu et al., 2020), animals, especially herbivorous insects (Chen et al., 2020), fungi (Torres-Farrada et al., 2017) and bacteria (Feng et al., 2015), and they may comprise of constitutive and inducible genes. The benefit of having such retinue of gene loci would be especially derived from applications beyond the spectrum of constitutively expressed laccase. Here, an operon switch would permit the seamless secretion and catalytic transformation of recalcitrant toxic wastes.

Close

Fig. 6

Gel representation (1.7 % w/v) of putative HRJ16 laccase genes amplified by PCR. Lane 1: molecular marker, lane 2: CueOP, lane 3: MCOStm, lane 4: CueOcit. The band sizes of laccase encoding genes can be observed with respect to the molecular marker and are as follows: CueOP; 156, 222, 261, 307, 559 and 745 bp; MCOStm; 303,653, 1025 and1483 bp; CueOcit;164, 339 and 542 bp.

Export to PPT

3.2 Biodecolorization and bioscouring capability of HRJ16 laccase

Astoundingly high concentrations (0.2 % w v^-1) of synthetic dyes of the heterocyclic cationic, triarylmethane, azo and anthraquinone groups were degraded over 80 h without the exogenous intervention of redox mediators (Fig. 7a). Specifically, AB was visibly decolorized from 16 h (ca. 49.3 %) of incubation up till terminal incubation period. This pattern differs markedly from the linear pattern it had portrayed in earlier studies (Unuofin, 2020b,c). Conversely, other synthetic dyes exhibited linear decolourization patterns, with marginal differences in decolorization occurring as incubation approached terminal stages. Navas et al. (2020), without the intervention of redox mediators, achieved increased decolorization of Methyl Orange at 60 °C over 24 h (ca. 50.95) under acid conditions (pH 5). Decolorization was minimal at neutral (pH 7: ca. 24.36) and alkaline conditions (pH 9: ca. 33.45); whereas only pH 5 was reported for effective decolorization of malachite green at 24 h (ca. 71.59). In another study, a 5 day treatment of Congo Red and Reactive Blue 4 at 37 °C elicited 16.43 % and 41.61 % decolorization, respectively (Liu et al. 2021); where a minimum decolourization was observed in BB, as only ca. 17.5 % decolourization was permitted. To our knowledge, the high substrate (dye) concentration might be responsible for the low levels of decolorization, which could have originated from a dead-end substrate-enzyme-intermediate complex. Moreover, steric hinderances that might be caused by the atomic orientation of the dyes could make laccase accessibility relatively unachievable. This, therefore, warrants further work, especially with regard to enzyme engineering, to enable the enzyme’s resilience and dexterity towards unorthodox substrates and their intermediates, as they may promote the degradation of heterogeneous toxic wastes. Reports regarding decolorization of high concentrations of dyes are gradually being documented, however, studies published by Bagewadi et al. (2017), Zhuo et al. (2017) and Kumari et al. (2018) are the closest coincidence to this present study.

Close

Fig. 7a

Decolourization pattern exhibited by HRJ16 laccase at 30 °C on 0.2 % synthetic dyes viz AB; azure B, MG; malachite green, RB; reactive blue, MO; methyl orange, CR; congo red, BB; brilliant blue. Phosphate buffer was used, with no mediator.

Export to PPT

Experiential dye tainting of the submerging medium was not drastic when only HRJ16 laccase were added to indigo fabric. However, a heterogeneous mix comprising HRJ16 laccase and a mediator (ABTS) (E + M) was sufficient to stimulate a progression of the medium’s colour from colourless to green, then indigo over 6 h (Fig S3). This began immediately after exogenous supply of TCA – a component of the laccase assay, which is suggestive of laccase oxidation of ABTS. Furthermore, the resultant indigo colour of the medium, which is the original pigment of the fabric evinced the empirical biobleaching of denim. A parallel experiment which had all other components of the reaction mixture, except laccase, ascertained the non-interference of TCA, since it is a potent bleaching agent. It could therefore be surmised that laccase biobleaching activity is experientially enhanced by an oxidized mediator, as corroborated by Solís-Oba et al. (2008). The aforementioned investigators through colorimetric assessment showed that the oxidized mediator-treated fabric had the brightest hue. Zhang et al. (2018) reported that a reaction mix comprising manganese peroxidase in malonate buffer with Mn²⁺ and 1-hydroxybenzotriazol (HBT: mediator), but without H₂O₂, could produce little or no biobleaching effects. Moreover, experiential bleaching effects were triggered when laccase was added to the mix. This phenomenon not only consolidates laccase’s status as an effective biobleaching agent but also reveals its potential as a vehicle for generating H₂O₂. In our study, a brighter hue was observed in fabrics that had undergone zymo-modification, however, it was brightest, post-treatment, with oxidized mediator (Fig. 7b Inset). This phenomenon was noted through colorimetric assessment by Solís-Oba et al. (2008), albeit there was no striking difference between their untreated and enzyme-modified fabric. This implies that HRJ16 laccase only requires oxidized mediators for acceleration of bleaching. Other mediators successfully applied include violuric acid (Iracheta-Cárdenas et al., 2016) and HBT (Yavuz et al., 2014). Consequently, other oxygenases might possess the denim bioscouring ability; this has been confirmed in a recent study (Zhang et al., 2020), where the singular and synergistic actions of manganese peroxidase and laccase (Zhang et al. 2022) produced enhance denim biobleaching at an acidic pH (4.8). This outcome coincided with Panwar et al. (2020), who observed improved reflectance at pH 4, but marginal or no reflectance at pH 8. With regard to reflectance, the constrast between the control (untreated) and the experimental (treated) is comparable to what was observed by Iracheta-Cárdenas et al. (2016), where the treated fabric recorded a higher reflectance reading (Fig. 7b). According to Tian et al. (2013) and references therein, Indigo dye is transformed by laccase oxidization, and it changes from its deep blue colour, through light blue to yield isatin (indole-2, 3-dione), a red substance, and further decomposed to anthranilic acid (2-aminobenzoic acid), which is a colourless substance. However, the spontaneous stepwise decolorization of the denim leachate, hours after the initiation of bleaching (Unuofin, 2020c) portrays a different colour transformation, though it completely transformed to a colourless solution. Although the mechanisms that govern this interesting outcome have not been satisfactorily ratiocinated, efforts are being made to understand the mechanism of this dual reaction (denim bleaching and dye decolorization). This is hoped to create a seamless dyeing of denims and the concomitant cleaning of dye baths. Moreover, intermediates formed during decolorization might serve as valuable feedstock for various industrial applications and should be characterized in future studies. The decolorization of denim leachates might not fix all the puzzles that riddle the textile industry, but it is indeed a major step towards achieving a circular biobased economy therein.

Close

Fig. 7b

Spectrum plot of Pseudomonas sp. HRJ16 laccase treated denim. HRJ16E3 represents enzyme only treatment, whereas HRJ16E3 + M3 represents enzyme + mediation treatment. Inset: experiential bleaching of denim fabric showing, from left to right: Untreated, HRJ16 laccase and HRJ16 laccase + mediator.

Export to PPT